Although the signals which direct splicing of messenger RNA precursors in vivo had been delineated, until the recent development of reliable in vitro systems, the mechanism by which intervening sequences are removed remained largely a mystery. Soon after the discovery of splicing a decade ago, it was postulated that U-class small nuclear RNAs, a group of metabolically stable capped RNAs ubiquitous in eukaryotes, were the factors responsible for decoding splicing signals. These models, based initially on hypothetical schemes for hydrogen- bonding with splicing substrates, appear to be broadly correct as judged by initial results with the cell-free systems. A great deal of refinement of the details is still necessary to bring the entire process into focus, however. Pathways such as splicing, in which an array of macromolecules assemble, are excellent targets for a genetic approach, but tractable genetic systems are distinctly lacking among the higher eukaryotes. The goal of this proposal is to exploit the powerful genetic methods available in yeast to achieve a precise understanding of the molecular contacts which contribute to accurate and efficient mRNA splicing. Schizosaccharomyces pombe has been chosen for our studies because, like Saccharomyces cerevisiae, it is amenable to genetic manipulation by both classical and transformation methods, but available evidence suggests that its splicing mechanism bears a closer resemblance to that of mammals. We are employing three complementary strategies. In the first, we will analyze null and conditional alleles of snRNA genes and determine their effects on RNA processing. Other components in the cell which interact with the snRNAs can then be identified by selecting for extragenic revertants of these mutations. A second approach will be to alter S. pombe splicing signals, which will converge with the snRNA work to allow definitive tests of proposed functional models, for example, hydrogen bonding between U1 snRNA and 5' splice junctions. The final goal which we will pursue is the development of an extract from S. pombe which splices mRNA precursors efficiently in vitro. The ultimate aim will be to use such a system to biochemically characterize mutations generated in the experiments described above.